Design of an ic-processed polymer nano-liquid chromatoraphy system on-a-chip and method of making it

ABSTRACT

Embodiments in accordance with the present invention relate to packed-column nano-liquid chromatography (nano-LC) systems integrated on-chip, and methods for producing and using same. The microfabricated chip includes a column, flits/filters, an injector, and a detector, fabricated in a process compatible with those conventionally utilized to form integrated circuits. The column can be packed with supports for various different stationary phases to allow performance of different forms of nano-LC, including but not limited to reversed-phase, normal-phase, adsorption, size-exclusion, affinity, and ion chromatography. A cross-channel injector injects a nanolitre/picolitre-volume sample plug at the column inlet. An electrochemical/conductivity sensor integrated at the column outlet measures separation signals. A self-aligned channel-strengthening technique increases pressure rating of the microfluidic system, allowing it to withstand the high pressure normally used in high performance liquid chromatography (HPLC). On-chip sample injection, separation, and detection of mixture of anions in water is successfully demonstrated using ion-exchange nano-LC.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application is a continuation of U.S. Ser. No. 12/111,159, filedApr. 28, 2008, which is a continuation of U.S. Ser. No. 10/917,257 filedAug. 11, 2004, which claims priority to U.S. Provisional patentapplication No. 60/496,964 filed Aug. 20, 2003, each of which areincorporated by reference in their entireties herein for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH OR DEVELOPMENT

Work described herein has been supported, in part, by the NSF CENSCenter (Grant No. CCR 0121778), the NSF ERC Center at the CaliforniaInstitute of Technology (Grant No. EEC-9402726) and the NationalInstitute of Health (grant No. R01RR06217). The United States Governmentmay therefore have certain rights in the invention.

BACKGROUND OF THE INVENTION

Separation of chemicals is widely and routinely performed in lots ofindustries and research labs. Liquid Chromatography (LC), and especiallyHigh Performance Liquid Chromatography (HPLC), is one of the mostpowerful and versatile separation techniques.

Although LC column is normally made of capillary tubes due to fluidicslimitations, the miniaturization of the column can actually improveseparation performance. As shown in FIG. 15 where same separationchemistry applies, separated peak width is independent of column ID,while peak heights are larger for smaller columns and/or smaller beads.

Conventional LC systems are also typically expensive and bulky. Theseparation columns, being of utmost importance in LC system, are alsoexpensive and need replacements after a certain times of usage(typically about 100 times). Sample and solvent consumption cost is alsovery high.

A miniaturized LC system could be cheaper, faster, and exhibit minimizedsample and solvent consumption. The need/market for miniaturized LCsystem is huge. However, comparing to the intense interests inminiaturized electrophoresis system on-a-chip, little is published aboutminiaturizing LC system onto a single chip. Harris et al., “Shrinkingthe LC Landscape”, Analytical Chemistry, pp. 64A-69A (February 2003),and de Mello, “On-chip chromatography: the last twenty years”, Lab on aChip 2, 48n-54n (2002), both of which are incorporated by referenceherein for all purposes, provide an overview of efforts in this area.

The main obstacles to miniaturization of LC systems are the lack of (1)a process to integrate various components of an LC system onto amonolithic chip; (2) high-pressure microfluidics needed for pumpingliquid through densely-packed beads column; and (3) an approach toeasily and reliably pack and seal chromatography supports (micro-beads)into the on-chip column.

From the above, it is seen that structures for performing liquidchromatography on small scales are highly desirable.

BRIEF SUMMARY OF THE INVENTION

Design and embodiments in accordance with the present invention relateto packed-column nano-liquid chromatography (nano-LC) systems integratedon-chip, and methods for producing and using same. The microfabricatedchip includes a column, frits/filters, an injector, and a detector,fabricated in a process compatible with those conventionally utilized toform integrated circuits. The column can be packed with supports forvarious different stationary phases to allow performance of differentforms of LC, including but not limited to reversed-phase, normal-phase,adsorption, size-exclusion, affinity, and ion chromatography. Across-channel injector injects a nanolitre/picolitre-volume sample plugat the column inlet. An electrochemical/conductivity sensor integratedat the column outlet measures separation signals. A self-alignedchannel-strengthening technique increases pressure rating of themicrofluidic system, allowing it to withstand the high pressure normallyused in nano-high performance liquid chromatography (nano-HPLC). On-chipsample injection, separation, and detection of mixture of anions inwater is successfully demonstrated using ion-exchange nano-LC.

An embodiment of method in accordance with the present invention forfabricating a liquid chromatography system, comprises, patterning asacrificial material on a first side of a substrate to define a columnregion, forming an encapsulant over the first side of the substrate andthe sacrificial material, and removing the sacrificial material todefine a column. Access is then provided to an inlet of the columnregion and to an outlet of the column region.

An embodiment of a liquid chromatography apparatus in accordance withthe present invention, comprises, a column defined between a substrateand a deposited Parylene layer, a column inlet in fluid communicationwith a first end of the column, and a column outlet in fluidcommunication with a second end of the column opposite the first end.

An embodiment of a method in accordance with the present invention forperforming liquid chromatography, comprises, introducing a liquid sampleat an inlet of a column defined between a deposited Parylene layeradhered to a substrate, flowing the sample down the column to an outlet,and detecting a changed property of the sample at the outlet.

Various additional features and advantages of the present invention canbe more fully appreciated with reference to the detailed description andaccompanying drawings that follow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1( a) shows a fluorescent top-view photograph of an embodiment ofan integrated nano-LC system in accordance with the present invention,including column, filter/flits, on-chip injector and detector.

FIG. 1( b) shows an optical photograph of the nano-LC device of FIG. 1(a) after packing beads into the column.

FIG. 2( a) illustrates a cross-sectional view of the nano-LC device ofFIGS. 1( a)-(b).

FIG. 2( b) shows a top view photograph of the nano-LC device of FIG. 2(a) including a packed column, a filter, sensing Pt/Ti electrodes, andchannel strengthening moat.

FIG. 2( c) shows a fluorescent picture of densely packed beads in anon-chip column.

FIGS. 3( a)-(h) show simplified cross-sectional views of one embodimentof a process for fabricating a nano-LC system in accordance with thepresent invention.

FIGS. 4( a)-(h) show simplified cross-sectional views of an alternativeembodiment of a process for fabricating a nano-LC system in accordancewith the present invention.

FIGS. 5( a)-(b) show cross-sectional views of Parylene channels madewith different anchoring techniques in accordance with embodiments ofthe present invention.

FIGS. 6( a)-(b) show cross-sectional photographs of a Parylene channelanchored with special trench with mushroom profile at the trench bottom.

FIG. 7 shows a top view photograph of a XeF₂ roughened channel moatbefore Parylene deposition.

FIG. 8 shows a photograph of the beads used to pack on-chip columns inaccordance with embodiments of the present invention.

FIG. 9 plots on-chip packed column flow rate versus pressuremeasurement.

FIG. 10 plots impedance frequency response of the conductivity detectioncell.

FIG. 11 shows ion-exchange liquid chromatography separation anddetection of five anions in water using integrated nano-LC systemon-a-chip.

FIG. 12 shows ion-exchange liquid chromatography separation anddetection of five anions in water using integrated nano-LC systemon-a-chip.

FIG. 13 shows ion-exchange liquid chromatography separation anddetection of seven anions in water using integrated nano-LC systemon-a-chip.

FIG. 14 shows separation of common anions using a commercialion-exchange column and commercial ion liquid chromatography system.

FIG. 15 shows separated peak shapes obtained with different column andbead size.

FIG. 16( a) shows an exploded view illustrating the parts of the testingjig.

FIG. 16( b) shows a photograph of the assembled package.

FIGS. 16( c)-(d) show simplified side views of the assembled package.

FIG. 16( e) shows a simplified plan view of the assembled package.

FIG. 16( f) shows a simplified perspective view of the assembledpackage.

FIGS. 16( g)-(i) show schematic plan views illustrating theconfiguration of holes on the jig in various orientations.

FIG. 17( a) shows a photograph of a microfabricated nano-LC column inaccordance with an embodiment of the present invention, exhibiting anearly-rectangular cross-sectional profile.

FIG. 17( b) shows a photograph of a microfabricated nano-LC column inaccordance with an embodiment of the present invention, exhibiting arounded cross-sectional profile.

FIG. 18 shows simplified plan view of an alternative embodiment of amicrofabricated nano-LC system in accordance with the present invention,utilizing on-chip electronic background suppression.

DETAILED DESCRIPTION OF THE INVENTION

As employed in this patent application, the term “nano-LC” refers to aliquid chromatography system employing a column inner diameter (ID) ofless than 100 μm. Where such a nano-LC system is employed with acorresponding flow rate on the order of tens to hundreds of nL/min, thesystem is referred to as a “nano-HPLC system”.

Embodiments in accordance with the present invention relate topacked-column nano-liquid chromatography (nano-LC) systems integratedon-chip, and methods for producing and using same. The microfabricatedchip includes a column, flits/filters, an injector, and a detector,fabricated in a process compatible with those conventionally utilized toform integrated circuits. The column can be packed with supports forvarious different stationary phases to allow performance of differentforms of nano-LC, including but not limited to reversed-phase,normal-phase, adsorption, size-exclusion, affinity, and ionchromatography. A cross-channel injector injects ananolitre/picolitre-volume sample plug at the column inlet. Anelectrochemical/conductivity sensor integrated at the column outletmeasures separation signals. A self-aligned channel-strengtheningtechnique increases pressure rating of the microfluidic system, allowingit to withstand the high pressure normally used in high performanceliquid chromatography (nano-HPLC). On-chip sample injection, separation,and detection of mixture of anions in water is successfully demonstratedusing ion-exchange LC.

1. Overview

FIG. 1( a) shows a fluorescent top-view photograph of one embodiment ofan integrated nano-LC system in accordance with the present invention.Integrated nano-LC system 100 comprises column 102, filter/flits 104,on-chip injector 106, and detector/sensor 108. FIG. 1( b) shows anoptical photograph of the nano-LC device of FIG. 1( b) after packingbeads 103 into the column 102.

FIG. 2( a) shows a cross-sectional view illustrating the nano-LC deviceof FIG. 1( a). FIG. 2( b) shows a top view picture of column 102 packedwith 7 μm ion-exchange beads 103, filter 104, sensing Pt/Ti electrodes108, and channel strengthening moat 110. FIG. 2( c) shows a fluorescentpicture of densely packed beads 103 in an on-chip column 102.

Backside holes 112 provide channel inlet and outlet. Beads 103 arepacked into the column 102 with the filters 104 at the column outlet tostop beads. The filter 104 holds beads in place because its openings aresmaller than the diameter of the beads. In FIG. 2( b), it can be clearlyseen that the beads 103 stop at the front of the filter 104.

The nano-LC-on-a-chip device of FIGS. 1( a)-(b) is made using integratedParylene microfluidics. LC stationary-phase support material comprisingmicro-beads with functional groups, is packed externally into theon-chip column from the inlet port located at point #1.

The separation mobile phase is also pumped from point #1 to point #2.The column is serpentine in shape to maximize column length within anarea of the chip occupied by the column. The beads-packed column outletis at the filter/detector at point #2, and the column inlet is near thefilter at point #4 to receive the injected plug front.

Nanolitre/picolitre volume sample injection is achieved withcross-channel injection from points #3 to #4. Such cross-channelinjection is described in detail by O'Neill et al., “On-chip Definitionof Picolitre Sample Injection Plugs for Miniaturized LiquidChromatography”, Journal of Chromatography A, 924, 259-263 (2001),incorporated by reference herein for all purposes.

The integrated design shown in FIGS. 1( a)-(b) minimizes dead volume,thus reducing extra-column peak broadening. Filters 104 and channels 102with height smaller than the bead's diameter, near points #3 and #4prevent the beads from entering the side channels.

To anchor Parylene channels, a “moat” surrounding channels is subjectedto deep reactive ion etching (DRIE) and/or roughened with XeF₂, thenfilled with Parylene. Fabrication of the moat is discussed below.

As shown in FIGS. 2( a)-(b), conductivity sensor 115 is used as thedetector for ion sensing. Interdigital electrodes 108 are patterned inthe detector cell 115 to monitor liquid conductivity. The conductivityof the mobile phase solution provides a baseline signal. When separatedion plugs pass by the detector, changes of solution conductivity aredetected. Chromatogram is then obtained by recording conductivity of thesolution flowing in the detection cell over time.

To increase sensitivity, electrode width and spacing should be small,and the total electrode area should be maximized. However, the electrodedesign should be compromised in considerations of minimizing peakbroadening and multiple-peak detection.

Off-chip pumping is used for mobile phase delivery and sample injection.On-chip pumps can be integrated if necessary. A detailed discussion ofintegrated on-chip gradient-generating micro-pumps is found in U.S.nonprovisional patent application Ser. No. 10/603,573, filed Jun. 24,2003 and incorporated by reference herein for all purposes.

2. Fabrication

FIGS. 3( a)-(h) show simplified cross-sectional views of one embodimentin accordance with the present invention of a process flow forfabricating the nano-LC device. As shown in FIG. 3( a), the fabricationprocess starts with growing thermal oxide 300 on both sides 302 a and302 b of a silicon wafer 302. Then two-step DRIE is done on the backside302 b to etch the backside holes 304, leaving only a 50 μm-thickdiaphragm 302 c.

FIG. 3( b) shows the following frontside oxide patterning, followed byevaporation and patterning of 300 Å Ti/2000 Å Pt/1000 Å Au electrodes306. Au is only for wire bonding purpose, since it is very difficult todirectly bond to Pt. Therefore, Au is patterned to be only at bondingpads. Pt is the electrode for electrochemical/conductivity sensing, andis patterned with hot Aqua Regia (1 HNO₃: 6 HCI: 3 H₂O at 80° C.). Ti isused as adhesion layer between Pt and substrate.

As shown in FIG. 3( c), 25 μm-thick photoresist AZ4620 is spun on thefront side of the substrate and patterned with two masks to formtwo-level sacrificial structure 308. Unexposed areas 308 a are forforming channels. Partially exposed areas 308 b are for forming filtersand/or for patterning oxide later. Fully exposed areas 308 c revealoxide 300 b underneath.

FIG. 3( d) shows the following stage in the fabrication process flow,wherein the oxide 300 b revealed by development of the filly exposedareas is subsequently etched away with BHF. Next, DRIE masked byphotoresist is performed on the front side to remove silicon and formtrench moat 309. Alternatively, or in conjunction with DRIE of theexposed silicon during this step, BrF₃/XeF₂ may be used to roughenexposed silicon and thereby promote adhesion of the Parylene depositedin the next step.

Next, as shown in FIG. 3( e), Parylene 310 is then deposited over theentire structure, including over the photoresist and within the moatarea. As shown in this and the previous figure, formation of the trenchmoat and/or roughened silicon regions is masked by the existingphotoresist, thereby constituting a self-aligned process. Theself-aligned nature of this process eliminates the need for a separatemasking step and enhances throughput.

In FIG. 3( f), the Parylene deposited in FIG. 3( d) is patterned to formchannels 312. Photoresist outside channels 312 is then stripped.

FIG. 3( g) then shows backside opening of access holes 314 with DRIE.Photoresist in the channels is then dissolved in Acetone, thus thechannels 312 are released.

Finally, as shown in FIG. 3( h), an optional SU-8 layer 316 could beapplied and patterned to further strengthen the channels.

FIGS. 4( a)-4(h) show simplified cross-sectional views of an alternativeprocess flow for forming a nano-LC system in accordance with embodimentsof the present invention. FIGS. 4( a)-(f) are identical to correspondingFIGS. 3( a)-(f). FIGS. 4( g)-(h), however, illustrate that the SU-8layer 316 can alternatively be applied before opening backside holes 314and removal of photoresist.

FIG. 16( a) shows an exploded view illustrating the parts of a packagingjig developed for convenient testing of the chips, and can be used as achip packaging method for real applications. FIG. 16( b) shows aphotograph of the assembled package.

Specifically, FIGS. 16( a)-(b) show that the fabricated chip is clampedbetween a printed circuit board (PCB) 1601 and the PEEK jig 1606.Squeezed o-rings 1600 at chip 1602 backside provide sealing. While thespecific embodiment of FIG. 16( a) includes o-rings as separate sealingelements, this is not required by the present invention. In accordancewith alternative embodiments, the sealing element could comprise apolymer gasket layer.

Standard fining receiving ports 1604 are made in the jig 1606, so fluidconnections with external sources are easily obtained. Electricalcontacts are made by wire bonding from the on-chip Au pads to the PCB.The jig can access eight out of the sixteen holes (4×4) simultaneouslyon the chip.

FIGS. 16( c)-(f) show side, top, and perspective views illustratingadditional details about the jig. The jig body 1606 is made of PEEKmaterial, which is inert for most chemicals and solvents. Commercialfitting receiving ports are machined in the jig as the interface betweenthe jig and the outside tubing with commercial fittings. Drilled holesin the jig lead fluid from the receiving ports to the jig top surface.

The jig top surface has two shapes of recesses. The square recess 1610is used for holding the chip. The circular recesses 1612 are foro-rings.

There are eight holes 1608 in the square recess, which connects with theeight receiving ports 1604 on the four sides of the jig 1606. The holes1608 are arranged so that by rotating the chip or jig by 90°, adifferent set of eight hole positions can be accessed. In this manner, atotal of sixteen hole positions in the form of a 4×4 array can beaccessed on the chip, with at most eight holes accessed simultaneously.

FIG. 16( g) shows the eight accessible hole positions in the originalorientation of the jig. FIG. 16( h) shows the location of another eighthole positions accessible after a rotation of the jig 90° from theorientation of FIG. 16( g). FIG. 16( i) shows a composite of the jigorientations of FIGS. 16( g)-(h), illustrating that a 4×4 array ofsixteen hole positions can be accessed, eight positions at a time.

As shown in the view of FIG. 16( b) depicting the assembled package,when the o-rings and chip are placed in their recesses, a top PCB(Printed Circuit Board) cover is placed on top and compresses chip ando-rings. The o-rings provide sealing between the chip and jig. Thenelectrical access to the chip is made by wire-bonding or even solderingfrom the chip to the top PCB.

Liquid chromatography system is known to be operating at very highpressures. Miniaturized on-chip nano-LC system can operate at much lowerpressure since the column cross-sectional area and column length couldbe smaller. However, it would still be desirable to have at least 100psi compatibility in order to perform High Performance LiquidChromatography (nano-HPLC) on-chip.

Parylene to substrate adhesion is poor, which is usually improved byapplying chemical adhesion layer (for example, A-174) on the substratebefore Parylene deposition. And Parylene-to-Parylene adhesion is usuallyenhanced by roughing the bottom Parylene with oxygen plasma.Nevertheless, even with these techniques, Parylene channels on substrateor on Parylene, can still withstand pressure up to only about 30 psi.Above that pressure, top Parylene layer would delaminate from substrateor from bottom Parylene.

This value may be compared with that exhibited by PDMS devices describedby Thorsen et al., in “Microfluidic Large-Scale Integration”, Science298: 580-584 (2002), incorporated by reference herein for all purposes,which are commonly used in microfluidic/μTAS area. Such PDMS devices canwithstand pressure only up to a bit over 40 psi.

In “Robust Parylene-to-Silicon Mechanical Anchoring”, The 16^(th) IEEEInternational Conference on Micro-Electro-Mechanical Systems, Japan, pp.602-605 (MEMS '03), incorporated by reference herein for all purposes,Liger et al. describe mechanical Parylene anchoring to the substrate. In“Parylene Neuro-Cages for Live Neural Networks Study”, 12th Intl Conf.on Solid-State Sensors, Actuators and Microsystems, Boston, pp. 995-998(Transducers '03), incorporated by reference herein for all purposes, Heet al. also describe the use of this technique to anchor neuro-cages tosubstrate, which allows the neuro-cages to survive aggressive chemicalsused in culturing experiments

These experiences lead to the invention of a specialchannel-strengthening technique has been invented in the above processesto strongly anchor Parylene channels to the substrate, to increasepressure rating of the system. Specifically, a self-alignedchannel-anchoring technique in accordance with embodiments of thepresent invention, uses a channel-surrounding moat that is self-alignedto the channel edges. The moat can be special trench into Si substratemade with modified DRIE process, or BrF₃/XeF₂ roughened Silicon.

FIGS. 5( a) and 5(b) illustrate cross-sectional views of finishedchannels made with these two anchoring techniques, respectively. Thetrench of FIG. 5( a) is made with standard DRIE Bosch process followedby short time SF₆ isotropic etching to create the mushroom profile atthe trench bottom.

FIG. 6( a) shows a photograph of a cross-sectional view of a Parylenechannel anchored with special trench with mushroom profile at the trenchbottom, 40 μm-deep. FIG. 6( b) shows an enlarged view of a portion ofthe cross-section shown in FIG. 6( a).

FIG. 7 is a top view photograph of an XeF₂ roughened moat surroundingpatterned photoresist (channel sacrificial material) before Parylenedeposition, for the second (channel roughening) strengthening approachshown above in FIG. 5( b).

3. Testing

To test the pressure limit of strengthened channels, a special testingchannel having only one access hole (inlet) is used. Water is droppedover the channel, and pressurized N₂ gas is applied to the channel. Oncethe channel is broken, bubbles will leak out and show up as bubbles inwater. Table 1 is the summary of the testing results.

TABLE I Channel Strengthening Technique Roughening + Trench + RougheningEpoxy Trench epoxy Safe Pressure  250 psi  700 psi  600 psi  800 psi(>60 min) (>60 min) (>60 min) (>60 min) Above Safe Small Bubbles SmallBubbles No Bubble N/A Pressure Appear Appear before Breaking Breaking~350 psi ~800 psi ~700 psi ~800 psi Pressure

The safe pressures obtained are well above desired 100 psi, and thehighest even reach 800 psi, which is limited by our testing setup.Embodiments of nano-LC systems in accordance with the present inventioncould be utilized with applied pressures of 1000 psi or greater in orderto perform high performance liquid chromatography on a chip.

Instead of SU-8, epoxy is used to further strengthen the channels, asSU-8 is one kind of photopatternable epoxy. It is clear that trenchanchoring technique provides superior anchoring performance toroughening. It should be noted that pressure limits obtained also dependon moat width, trench depth/shape, and parylene thickness.

The failure modes of the two anchoring techniques are also different.Roughening-anchored channels leak out working fluid through tiny tunnelsin roughened moat area under high pressure, while trench-anchored onesdon't leak at all until the Parylene breaks.

In terms of introduction and retaining nano-LC stationary phasematerials into the column, several approaches have previously beenproposed. In “Design of an open-tubular column liquid chromatographyusing silicon chip technology”, Sensors and Actuators B1 249-255 (1990),Manz et al. describe an open-tubular approach. In “Fabrication ofnanocolumns for liquid chromatography”, Anal. Chem. 70, 3790-3797(1998), He et al. describe coating micromachined posts arrays. In “Ionchromatography on-chip”, J. of Chromatography A, 924, 233-238 (2001),Murrihy et al. et al describe coating a microchannel with nanoparticles.In “Gradient-elution reversed-phase electrochromatography inmicrochips”, Proc. μTAS 2003, pp. 1163-1166, Singh et al. describe amonolithic device. In “High performance liquid chromatography partiallyintegrated onto a Silicon chip”, Analytical Methods and Instrumentation,Vol. 2 No. 2, 74-82 (1995), Ocvirk et al describe a packing approach. In“An integrated fritless column for on-chip capillaryelectrochromatography with conventional stationary phases”, Anal. Chem.,2002, 74, 639-647, Ceriotti et al. describe an approach utilizingpacking without a fit. Each of the above-referenced publications ishereby incorporated by reference for all purposes.

In accordance with embodiments of the present invention, a slurrypacking technique is used to pack LC stationary phase support material(beads) into on-chip columns. First the beads are mixed with water orIsopropyl Alcohol, then a vortexer is used to homogenize the solution,also to prevent beads from precipitation. Then the solution is suckedinto a syringe/pipette, and injected into a section of tubing as a beadssolution reservoir. The tubing fill of beads/solution mixture is thenswitched on-line, and off-chip pressure source is used to pack the beadsinto the column on-chip. The pressure source can be simply pushingsyringe manually, or a syringe pump or pressurize gas.

To get a uniform and dense packing, constant high pressure is preferred.The columns we used to do testing are all packed at 200 psi usingpressurized N₂ gas. After the beads are packed, the beads bed won't flowback even when the inlet pressure is released.

In this work, packing with conventional beads is chosen because of thefollowing advantages. First, without introducing new surface chemistry,the extensively established separation knowledge can be utilized.Secondly, extreme flexibility is there to perform different types ofliquid chromatography and/or to optimize particular separations, bychoosing bead type, size, pore size, porosity, and functional group.Thirdly, packed column can achieve reproducible column performance,which is usually a problem in other methods.

FIG. 8 shows one type of the beads used for packing and testing. Thebeads of FIG. 8 are 7 μm-diameter anion exchange resin(PolyStyrene-Divinylbenzene beads with Trimethyl-Ammonium groups) fromthe Hamilton Company of Reno, Nev. The resins are the same as the resinsin Hamilton's widely-used anion exchange column PRP-X110. FIG. 2( c)shows a section of on-chip column packed with the beads shown in FIG. 8.

Flow rate versus pressure curve is obtained after column is packed at200 psi. The sample injection inlet (#3) and outlet (#4) are sealed withepoxy before measurement. DI water is pumped through column underconstant pressure and flow rate is measured at column outlet bymeasuring liquid front moving speed in a capillary.

The volumetric flow rate is plotted against corresponding pressure inFIG. 9. It can be seen that the packed column provides huge backpressure, as the flow rate through the on-chip column is only about 3μl/min even at 200 psi.

According to the theory set forth by Meyer, Practical High PerformanceLiquid Chromatography, John Wiley & Sons, pp. 310-311(1999),incorporated by reference for all purposes herein, the followingequation governs volumetric flow rate:

${F = {\frac{ɛ}{\Phi}\frac{d_{p}^{2}A_{c}}{\eta \; L_{c}}\Delta \; P}};$

where

-   -   F=volumetric flow rate;    -   ΔP=column pressure drop;    -   ∈=porosity of packed column;    -   η=viscosity of the fluid;    -   d_(p)=beads diameter;    -   Φ=dimensionless flow resistance;    -   A_(c)=column cross-sectional area; and    -   L_(c)=column length.

From the fitted linear curve of FIG. 9, it is found that Φ/∈=444.Assuming the porosity of the packed column is 0.8, which is normal fordensely-packed porous-spherical-beads column, then Φ is 355, which isclose to but smaller than its empirical value of 500 for slurry packedspherical porous-beads in conventional LC columns. It is believed thatthis is partly due to the small on-chip column to bead size ratio.

Cross-channel injection method described above is used for injectingnanolitre/picolitre-volume samples into the column. Basically, thismethod limits flow path by controlling valves connected to the accessholes of the system. During normal mobile phase flow, the valvescontrolling flow through points #3 and #4 (FIG. 1( a)) for the sideinjection channels are closed. During injection, the valve controllingflow through points #1 and #2 for the main flow path are closed, and thevalves controlling flow through points #3 and #4 are open. Sample isinjected from #3 to #4. The injected sample plug size is determined bythe distance between the side channels, so that nanolitre-volume or evenpicolitre sample injection is easily achieved. Detailed discussion ofthe method can be found in the O'Neill et al publication incorporated byreference above.

The valves can be configured in different ways to perform variousfunctions, some of which are summarized in Table 2.

TABLE 2 CONFIGURATION OF VALVE AT POINT # #1 #2 #3 #4 Packing O O X XSample Flush X X O O Mobile Phase Flush O O X X Sample Injection X X O OElution O O X X Flush Column O O X/O X/O Flush Injection Loop O X O O

Interdigitated Pt/Ti electrodes are used in the on-chip detection cell,which is located immediately after the on-chip chromatography column.The electrodes are used to monitor solution conductivity. When separatedsample peaks flow through the detection cell, fluctuations in solutionconductivity are recorded. AC signal is used for measuring conductivity.To determine an optimal frequency, the impedance frequency responsecurve is measured for the mobile phase solution.

FIG. 10 shows impedance frequency response of the conductivity detectioncell. The curve indicates that above about 10 kHz, the impedance isalmost independent of frequency. Per Bohm et al., “A Closed-loopControlled Electrochemically Actuated Micro-dosing System”, Journal ofMicromechanics and Microengineering, 10 498-504 (2000), incorporated byreference herein for all purposes, it is adequate forconductivity-measuring frequency to be in this resistance dominantregion to minimize electrochemical effects.

To demonstrate on-chip liquid chromatography separation, ion-exchange LCis used partly because it is easy to verify separation using on-chipconductivity sensor. Anion mixtures in water with known concentrationshave been successfully separated and detected with the on-chipconductivity sensor.

The on-chip column had a length of 8 mm, a width of 100 μm, and a heightof 25 μm. The column was packed with PRP-X110 anion exchange 7 μm resin.Standard 1.7 mM NaHCO₃ and 1.8 mM Na₂CO₃ (Alltech Inc.) is used asmobile phase pumped at 0.2 μl/min isocratic with conductivity sensing.The injection volume is from 3 nl to tens of nanolitres. Thechromatograms of FIGS. 11-13 show the typical ion exchange separationresults.

Specifically, FIG. 11 shows ion-exchange liquid chromatographyseparation and detection of five anions in water using integrated LCsystem on-a-chip. The chromatogram of FIG. 11 was obtained using thebasic measuring circuit. FIG. 12 shows ion-exchange liquidchromatography separation and detection of the same five anions in waterwith the integrated nano-LC system on-a-chip, using the basic measuringcircuit plus lock-in amplifier.

Qualitative determination of the anions can be done by comparing thepeak elution time or retention factors to those of the standardchromatogram. Or if one of the peaks can be confirmed by other methods,for instance electrochemical technique, then other peaks can be deduced.

Quantitative determination of the sample concentrations can be obtainedby comparing the peak height/area to standard calibration curve, wherethe peak height/area is plotted against samples concentrations. Thisplot is typically linear. Table 3 shows a typical peak-analysis resultof the chromatogram shown in FIG. 12, which includes integrated peakareas, peak-area percentage, peak center positions, peak heights andpeak resolutions with respect to next peak.

TABLE 3 Area Area Resolution Peak Integra- Percent- Peak Peak w/Next #Anion tion age Center Height Peak 1 Fluoride 10.77885 52.19% 8.62211.7984 7.20554 2 Chloride 1.24881 6.05% 22.672 1.58945 14.72439 3Nitride 1.12842 5.46% 51.435 1.0591 24.99618 4 Bromide 5.54006 26.82%97.713 1.09348 20.45866 5 Nitrate 1.95858 9.48% 135.858 3.01061 —

FIG. 13 shows the result of ion-exchange liquid chromatographyseparation and detection of seven anions in water using integrated LCsystem on-a-chip using the basic measuring circuit only. The results ofFIG. 13 may be compared with FIG. 14, which shows separation of the sameseven common anions using the commercially available PRP-X110Sion-exchange column from Hamilton Corp. having an inner diameter of 4.1mm and a length of 150 mm. The beads used in this commercial column arethe same used in the on-chip column separations.

To summarize, main obstacles previously encountered in miniaturizing LCsystems include the lack of (1) a process to integrate variouscomponents of an LC system onto a monolithic chip; (2) high-pressuremicrofluidics needed for pumping liquid through densely-packed beadscolumn; and (3) an approach to easily and reliably pack and sealchromatography supports (micro-beads) into the on-chip column.

Various embodiments in accordance with the present invention address allof the three problems. Integrated Parylene microfluidics technology isused to fabricate the device. Channel-strengthening techniques areinvented to fulfill high-pressure requirements. Slurry technique isemployed to pack beads externally into the on-chip column. U.S.nonprovisional patent application Ser. No. 10/391,122, incorporatedherein by reference for all purposes, describes an alternative methodfor integrating beads into microfabricated columns using batchfabrication process.

The invented chip is an integrated Parylene microfluidic system. Thefabrication process is compatible with previously developed otherParylene microfluidic components, which can be integrated with thecurrent chromatography system to provide more powerful liquidmanipulation capability and also more separation and detection options,for example gradient separation and mass spectrometry detection.

The previously developed components include at least the following:gradient-generating electrolysis micro-pump, electrolysis micro-pump,peristaltic micro-pump, electro-spray nozzle, micro-check valve, microin-channel check valve, pressure sensors, flow sensors and shear stresssensor. These previously-developed components are described in thefollowing reference list.

Xie, et al., “Integrated Parylene Electrostatic Peristaltic Pump”, 7thInt'l. Syrup. on Micro Total Analysis System, California (μTAS 2003).Shih et al., “Surface Micromachined and Integrated Capacitive SensorsFor Microfluidic Applications”, 12^(th) Int'l. Conf. on Solid-StateSensors, Actuators and Microsystems, pp. 388-391, Boston (Transducers2003). Meng et al., “A Parylene MEMS Flow Sensing Array”, Transducers2003; Xie et al., “Electrolysis-Based On-Chip Dispensing system forESI-MS”, 16th IEEE Int'l MEMS Conf., Japan pp. 443-446 (MEMS '03). Xieet al., “Integrated Surface Micromachined Mass Flow Controller”, (MEMS2003). Meng et al., “A MEMS Body Fluid Flow Sensor”, California (μTAS2001). Xie et al., “Surface Micromachined Leakage Proof Parylene CheckValve”, 14th IEEE International Conference on MicroElectroMechanicalSystems, Switzerland, pp. 539-542 (MEMS '01). Wang et al., “A NormallyClosed In-Channel Micro Check Valve”, 13th IEEE International Conferenceon MicroElectro Mechanical Systems, Japan (MEMS '00). Wang et al, “AParylene Micro Check Valve”, 12th IEEE International Conference on MicroElectro Mechanical Systems (MEMS '99). Wang et al., “A Fully IntegratedShear Stress Sensor”, 10th Int'l Conf. on Solid-State Sensors, Actuatorsand Microsystems (Transducers '99). Each of these references isincorporated by reference herein for all purposes.

Furthermore, the fabrication processes in accordance with embodiments ofthe present invention are also compatible with post-CMOS IntegratedCircuits (IC) processes. In particular, the temperature duringfabrication typically does not exceed 200° C. By remaining comfortablybelow the 600° C. threshold temperature resulting in the melting ofinterconnect metals such as aluminum, potentially the whole testingcircuit, signal conditioning and processing, and even wirelesstransmission and power generation, could be integrated on thenano-liquid chromatography chip.

The on-chip column can be packed with any desired chromatographysupports. Therefore, all kinds of LC can be done with the invented chip.Since the chip has on-chip injector and detector, fittings and tubingconnecting injector and column, column and detector, are eliminated. Theinjected plug is right at the column inlet, and the separated componentsenter the detector right at the column outlet, which minimizesextra-column band broadening. Using on-chip injector, nanolitre or evenpicolitre size injection is easily achieved, which is extremelydifficult for conventional off-chip injection methods.

The micro liquid chromatography chip is made using batch fabricationprocess, which reduces its cost dramatically, making disposable nano-LCchip possible. The solvent/sample consumption is also loweredconsiderably. High throughput separations can also be done by integratedmultiple columns on-chip, as every column only takes small spaceon-chip. By packing the columns with different LC supports, varioustypes of nano-LC can be done on-chip simultaneously.

The foregoing discussion of the invention has been presented for purposeof illustration and description. The foregoing is not intended to limitthe invention to the form or forms disclosed herein. Possible variationsof the system include, but not limited to the following alternativedescribed embodiments.

Fabrication of the device is not limited to the specific embodimentsdescribed above in connection with FIGS. 3( a)-(h) and 4(a)-(h). Forexample, for faster fabrication, KOH or a combination of KOH and DRIEcan be used to etch backside holes.

Moreover, in the process flows of FIGS. 3( a)-(g) and 4(a)-(g), thebackside two-step access holes are used as a tubing stopper if tubing isto be directly inserted into the holes and bonded to chip backside. If,however, other packaging methods are used such as an O-ring seal, then astraight DRIE hole would be sufficient.

Moreover, the channel access holes are not required to be positioned onthe device backside, but could alternatively be positioned on the devicefrontside. In such an alternative embodiment, holes to gain access tothe channel portions could be created by punching or etching through thefront-side Parylene layer, and also through any overlying passivationlayer

And while the filter has been shown as being fabricated by partialexposure, this is not required by the present invention. Othertechniques may be utilized to hold the beads by restricting at least onedimension (width and/or height) in the flow path, and still remainwithin the scope of the present invention. One example is thefabrication of an array of posts to form a column frit/filter.

Moreover, while the specific process flow described above has utilizedParylene and photoresist as structural and sacrificial materials,respectively, other materials can be utilized to fabricate the channels.In addition, the substrate can be formed from other materials as well,including but not limited to silicon, glass, Pyrex, quartz, fusedsilica, polymer, and silicon-on-insulator (501).

And while the above description relates to the use of SU-8 as apassivation material, this is also not required by the presentinvention. In accordance with alternative embodiments, simple epoxy orother types of coating materials can be applied on top of the device tostrengthen the channels and to provide passivation.

In accordance with still other embodiments of the present invention,dimensions of different features of the device, such as the shape,height, width, or length of the channel, can be adjusted for differentapplications. The filter height can also be varied for use withdifferent bead sizes. Moreover, the configuration or size of theinjection channels can also be adjusted.

In accordance with still other embodiments of the present invention,variation on the manner of sample injection may be employed. Inaccordance with alternative embodiments the sequence of operation of thevalves can be changed, in order to achieve different injectionperformance. In accordance with still other alternative embodiments,different on-chip injection methods can also be used, for example,on-chip electrolysis-based sample injection.

In accordance with still other embodiments of the present invention, thecolumn can be packed utilizing methods other than the slurry techniquedescribed above. In some embodiments, an open column can be used insteadof a packed column. Such an open column can be coated withchromatography stationary phase materials as a separation support.

Various types of pumping methods can be used, including but limited tooff-chip pumping with pressurized gas or a syringe pump, on-chippressure-driven pumping, and/or electrically-driven pumping.Incorporated by reference herein for all purposes is U.S. nonprovisionalpatent application Ser. No. 10/391,122, incorporated by reference above,describes an on-chip electrolysis-based gradient pump capable ofperforming isocratic/gradient LC separations.

A variety of types of different devices may be in fluid communicationwith the outlet of the column, and remain within the scope of thepresent invention. For example, an electrospray nozzle can be integratedat the column outlet to couple the chip with Mass Spectrometer. Otherdetection methods can also be employed, for example, optical detectionsuch as UV or fluorescence, can be performed in the detector cell. Andsince the fabrication process is post-CMOS compatible, variousdetecting, measuring, signal processing, and transmission circuits canreadily be integrated on-chip.

In accordance with still further alternative embodiments of the presentinvention, the cross-sectional profile of the column may be determinedby the particular fabrication process. Specifically, LC separationperformance depends on the packing quality of the beads or otherstationary phase within the column. A column having a roundedcross-sectional profile is generally preferred to one exhibiting arectangular profile, as such a rectangular profile the latter leavesmore dead volume near the inner side of the column when packed withspherical LC beads. Since microfabricated columns have an even smallercolumn ID to bead diameter ratio, this effect could be more pronouncedfor nano LC columns. Therefore, under certain circumstances it may bedesirable to fabricate micromachined columns having roundedcross-sections.

Conversely, in other applications, a rectangular micromachined column,or a column exhibiting some combination of the rounded and rectangularcross-sectional profile, may be desired. For example, microfabricateddevices frequently employ optical detection based upon the simplicityand sensitivity of this sensing technique. With microfabricated nano-LCcolumns exhibiting circular cross-sections, light incident for detectionpurposes may be undesirably deflected when passing through the columnwall. Such unwanted optical deflection would not occur formicrofabricated columns exhibiting a cross-sectional profile that iswholly rectangular, or partially rectangular at the location oftransmission of the optical sensing beam.

Therefore, in an alternative embodiment of a fabrication method inaccordance with the present invention, the nano-LC column may betailored to exhibit a cross-sectional or rectangular profile fordifferent applications. Specifically, during formation of the column,sacrificial material can formed in a rounded cross-sectional profile.

The basic idea utilizes a thermal reflow property of a sacrificialmaterial, typically a photoresist. The sacrificial material used todefine the column can be thermally reflowed after photo-patterning (FIG.3( c) or 4(c)), but before deposition of the Parylene (FIG. 3( e) or4(e)). The higher the temperature and/or the longer the baking, the morerounded the cross-section will be obtained. To demonstrate the effect,two nano-LC columns with different cross-sections, one nearlyrectangular and the other nearly circular, were fabricated.

FIG. 17( a) shows a photograph of a nano-LC column microfabricatedwithout thermal reflow and exhibiting a nearly rectangularcross-sectional profile. FIG. 17( b) shows a photograph of anothernano-LC column microfabricated to have a rounded cross-section bythermal reflow of its column sacrificial photoresist during processingbefore parylene deposition. FIGS. 17( a)-(b) illustrate that thecross-sectional profile of the nano-LC column can be tailored fordifferent applications.

In accordance with still another alternative embodiment of the presentinvention, the sensitivity of the detector can be improved utilizingbackground suppression integrated on-chip. Specifically, a conductivitydetector records a baseline solution conductivity during elution of theliquid phase, when no sample ion is passing by. The conductivitydetector will detect a change of conductance when a sample ion is elutedfrom the column and enters the detector cell. This detected conductivitychange will be manifested as a peak or valley superimposed on thebaseline signal. The magnitude of this conductivity signal isproportional to the difference in conductance between the total elutedsolution and the background, as expressed in the following equation:

${\Delta \; G} = {{G_{Elution} - G_{Background}} = {\frac{1000}{K_{cell}}( {\lambda_{s^{-}} - \lambda_{E^{-}}} )C_{s}}}$

-   G_(Elution): Total conductance of the eluted solution-   G_(Background): Background conductance of the eluent-   K_(cell): Cell constant of the conductivity sensor-   λ_(S) ⁻ : Equivalent conductance constant for sample ion-   λ_(E) ⁻ : Equivalent conductance constant for eluent ion-   C_(S): Concentration of the sample ion to be detected

Normally, the peak of the conductance signal lies only a few percentover the background signal. This is especially true for ppm(pan-per-million) and ppb (part-perbillion) level sensing. Moreover, theeluent used in ion liquid chromatography typically exhibits a highconductivity, making the detection of low-concentrations of Ionsdifficult.

Conventionally, this sensitivity issue has been addressed by introducinga chemical suppression device between the outlet of the separationcolumn and the inlet of the detector. Such a chemical suppression devicereacts with the solution eluted from the column, reducing itsconductivity to nearly zero prior to entry into the detector. Byperforming this conventional background suppression, the detection limitof the LC system can usually be lowered by about three orders ofmagnitude.

An alternative embodiment in accordance with the present inventionintegrates on-chip an electronic suppression method that will remove thebackground conductance from the total signal. FIG. 18 shows a simplifiedschematic plan view of such an embodiment of a nano-LC system inaccordance with the present invention.

Nano-LC system 1800 of FIG. 18 comprises microfabricated column 1802having inlet 1802 a and outlet 1802 b. Nano-LC system 1800 furthercomprises two conductivity detectors having the same structure formedon-chip at different locations. First conductivity detector 1804 a islocated upstream of the point of sample introduction,

and is configured to measure background conductance only. Secondconductivity detector 1804 b is located downstream of the column outletand measures the total conductance of the solution eluted from column,including both sample and solvent.

Both the first and second conductivity detectors 1804 a and 1804 b arein electrical communication with comparator 1806. Comparator 1806comprises input nodes 1806 a and 1806 b, configured to receive signalsfrom detectors 1804 a and 1804 b, respectively. Comparator 1806 isconfigured to output at node 1806 c an amplified signal reflecting thedifference between the two sensors' response.

An embodiment of a nano-LC system utilizing background suppressionoffers a number of potential benefits. One such benefit is the effectivesuppression of the background signal, allowing the detection limit to belowered by several orders of magnitude.

Another potential benefit is ease of fabrication. Specifically, a systemincluding the additional conductivity sensor can easily be integratedwithin an existing fabrication scheme such as is shown in FIG. 3( a)-(h)or 4(a)-(h), requiring only changes to the mask layout without requiringadditional steps that could result in lower throughput and increasedexpense.

Further variation on specific embodiments of the present invention shownand discussed so far are possible. For example, an alternativeembodiment in accordance with the present invention could utilize a flowpath comprising multiple successive columns employing different packingmedia or separation chemistry to perform multi-dimensional separations.Still other alternative embodiments could utilize a flow path comprisingmultiple parallel columns employing different packing media orseparation chemistry to simultaneously perform multiple separation ofthe components of multiple samples. In accordance with still otherembodiments, a single chip could include a plurality of columnsconfigured to perform separations to isolate components of multiplesamples, and also to conduct multi-dimensional separation to isolatecomponents of a single sample.

The small size of embodiments of nano-LC devices in accordance with thepresent invention, coupled with their ability to performchemical/biological sensing on-chip, renders them suitable for use in avariety of applications. In particular, employment of componentseparation techniques upstream of detection, reduces or eliminates theneed to provide specialized detection devices of limited applicabilityto only certain target materials. Thus nano-LC devices in accordancewith embodiments of the present invention are suitable for uses such asmonitoring of the environmental condition of remote sites, networkedsensing, and consumer health care.

It is to be understood that the examples and embodiments describedherein are for illustrative purposes only, and there can be othervariations and alternatives. Various modifications or changes in lightof the above description thereof will be suggested to persons skilled inthe art and are to be included within the spirit and purview of thisapplication and scope of the appended claims.

1. A method of fabricating a nano-liquid chromatography systemon-a-chip, the method comprising: patterning a sacrificial material on afirst side of a substrate to define a column region; forming anencapsulant over the first side of the substrate and the sacrificialmaterial; removing the sacrificial material to define a column; andproviding access to an inlet of the column region and an outlet of thecolumn region.
 2. The method of claim 1 wherein providing access to thecolumn inlet and the column outlet comprises etching through a backsideof the substrate to stop on the sacrificial material.
 3. The method ofclaim 1 wherein providing access to the column inlet and the columnoutlet comprises forming a hole through the encapsulant present in afront side of the substrate.
 4. The method of claim 1 wherein:patterning the sacrificial material comprises developing a resistmaterial utilizing lithography; and removing the sacrificial materialcomprises stripping the developed resist material.
 5. The method ofclaim 4 wherein patterning the sacrificial material further comprisespartially developing the resist material to define a constriction at thecolumn outlet.
 6. The method of claim 4 wherein the constrictioncomprises an opening narrower than a column packing material.
 7. Themethod of claim 4 wherein patterning the sacrificial material furthercomprises patterning a post to define a constriction at the columnoutlet.
 8. The method of claim 4 further comprising reflowing thedeveloped photoresist to form a rounded cross-sectional profile.
 9. Themethod of claim 1 wherein forming the encapsulant comprises depositingParylene.
 10. The method of claim 9 wherein prior to deposition of theParylene, a moat is etched into the substrate adjacent to the channel toreceive and anchor the deposited Parylene.
 11. The method of claim 10wherein the moat etching is self-aligned to the sacrificial material.12. The method of claim 9 wherein prior to deposition of the Parylene,regions adjacent to the channel are roughened by chemical exposure toenhance adhesion with the deposited Parylene.
 13. The method of claim 12wherein the roughening is self-aligned to the sacrificial material. 14.The method of claim 1 further comprising forming a passivating layerover the encapsulant.
 15. The method of claim 14 wherein the passivatinglayer is formed subsequent to removal of the sacrificial material. 16.The method of claim 14 wherein the passivating layer is formed prior toremoval of the sacrificial material.
 17. The method of claim 1 furthercomprising patterning a conducting electrode on the substrate proximateto an expected outlet of the column, prior to patterning the sacrificialmaterial.
 18. The method of claim 1 wherein the sacrificial material isalso patterned to form a sample injector region intersecting the columnregion, the method further comprising providing access to an inlet ofthe injector region and to an outlet of the injector region.
 19. Themethod of claim 1 wherein a temperature during fabrication does notexceed 200° C.
 20. The method of claim 1 further comprising introducinga packing material into the column.
 21. A nano-liquid chromatographyapparatus on-a-chip comprising: a column defined between a substrate anda deposited Parylene layer; a column inlet in fluid communication with afirst end of the column; and a column outlet in fluid communication witha second end of the column opposite the first end.
 22. The nano-liquidchromatography apparatus of claim 21 wherein the column is serpentine inshape.
 23. The nano-liquid chromatography apparatus of claim 21 whereinthe deposited Parylene layer remains adhered to the substrate atpressures of 1000 psi or greater within the column.
 24. The nano-liquidchromatography apparatus of claim 21 wherein the substrate includes aroughened region adjacent to the column and configured to promoteadhesion between the deposited Parylene layer and the substrate.
 25. Thenano-liquid chromatography apparatus of claim 24 wherein the roughenedregion is self-aligned to the column.
 26. The nano-liquid chromatographyapparatus of claim 21 wherein the substrate defines a moat adjacent tothe column and configured to receive the deposited Parylene layer andpromote adhesion between the deposited Parylene layer and the substrate.27. The nano-liquid chromatography apparatus of claim 21 wherein themoat is self-aligned to the column.
 28. The nano-liquid chromatographyapparatus of claim 21 further comprising a column outlet having aconstriction narrower than a column packing material.
 29. Thenano-liquid chromatography apparatus of claim 21 further comprising acolumn outlet having a constriction configured to cause the outlet to bejammed with packing material during column loading.
 30. The nano-liquidchromatography apparatus of claim 21 further comprising an injectorintersecting the column inlet.
 31. The nano-liquid chromatographyapparatus of claim 21 further comprising a detector positioneddownstream the column outlet.
 32. The nano-liquid chromatographyapparatus of claim 31 wherein the detector is positioned immediatelyadjacent to the column outlet to minimize an intervening dead volume.33. The nano-liquid chromatography apparatus of claim 31 wherein thedetector comprises an electrode patterned on the substrate.
 34. Thenano-liquid chromatography apparatus of claim 31 further comprising: asecond detector positioned upstream of the column inlet; and acomparator in communication with the detector and the second detector,the comparator configured to output a signal reflecting a differencebetween the detector and the second detector.
 35. The nano-liquidchromatography apparatus of claim 21 wherein the column exhibits arounded cross-sectional profile.
 36. The nano-liquid chromatographyapparatus of claim 21 further comprising a second column defined betweenthe substrate and the deposited Paraylene layer, an inlet of the secondcolumn in fluid communication with the first column outlet, the firstand second column configured to perform multi-dimensional separation ofcomponents of a sample.
 37. The nano-liquid chromatography apparatus ofclaim 21 further comprising a second column defined between thesubstrate and the deposited Paraylene layer, the first column inlet andan inlet of the second column in fluid communication with a commoninlet, the first and second column configured to simultaneously performmultiple separation of components of a sample introduced to the commoninlet.
 38. The nano-liquid chromatography apparatus of claim 21 furthercomprising a packaging jig including: a first surface defining an outletport, the first surface configured to receive the substrate and placethe outlet port in fluid communication with the column inlet; a secondsurface defining an inlet port configured to receive a liquid from anexternal source; and a body including a conduit configured to place theinlet port in fluid communication with the outlet port.
 39. Thenano-liquid chromatography apparatus of claim 38 further comprising asealing element positioned between the first surface and the substrate.40. The nano-liquid chromatography apparatus of claim 39 wherein thesealing element comprises an 0-ring.
 41. The nano-liquid chromatographyapparatus of claim 39 wherein the sealing element comprises a polymergasket layer.
 42. The nano-liquid chromatography apparatus of claim 38wherein positioning the substrate on the first surface at a differentorientation relative to the packaging jig places the outlet port influid communication with a different chip inlet.
 43. The nano-liquidchromatography apparatus of claim 38 further comprising a printedcircuit board in contact with a side of the substrate opposite thepackaging jig, a conducting contact on the printed circuit board inelectrical communication with a detector.
 44. The nano-liquidchromatography apparatus of claim 21 further comprising a column accesshole formed through the Parylene on a chip front side and in fluidcommunication with the column inlet.
 45. The nano-liquid chromatographyapparatus of claim 21 further comprising a column access hole formedthrough the substrate on a chip back side and in fluid communicationwith the column inlet.
 46. A method of performing nano-liquidchromatography comprising: providing at an inlet of a column definedbetween a deposited layer adhered to a substrate, a sample including aplurality of components; flowing a mobile phase down the column toseparate the plurality of sample components; and detecting a changedproperty at a column outlet to reveal elution of one of the plurality ofsample components.
 47. The nano-liquid chromatography method of claim 46wherein the sample is provided to a column outlet defined between adeposited Parylene layer adhered to the substrate.
 48. The nano-liquidchromatography method of claim 46 wherein the sample is provided to theinlet by cross-flow injection.
 49. The nano-liquid chromatography methodof claim 46 wherein the mobile phase is flowed down the column at apressure of 1000 psi or greater.
 50. The nano-liquid chromatographymethod of claim 46 wherein the mobile phase is flowed down the column ata pressure of less than 1000 psi.
 51. The nano-liquid chromatographymethod of claim 46 wherein a changed conductance is detected at anelectrode present on the substrate at the column outlet.
 52. Thenano-liquid chromatography method of claim 46 further comprisingcomparing a sensed property of the mobile phase at the column inlet witha sensed property of the component at the column outlet.
 53. Thenano-liquid chromatography method of claim 46 further comprising placingthe column inlet in fluid communication with an external source via aninlet port located on a first surface of a packaging jig, the inlet portin fluid communication with the column inlet through an internal jigconduit and an outlet port on a second surface of the packaging jig. 54.The nano-liquid chromatography method of claim 53 further comprisingsealing the column inlet to the outlet port with an o-ring.
 55. Thenano-liquid chromatography method of claim 53 further comprising sealingthe column inlet to the outlet port with a polymer gasket layer.
 56. Thenano-liquid chromatography method of claim 53 further comprisingestablishing electronic communication with a detector on the substratethrough a printed circuit board in contact with a side of the substrateopposite the packaging jig.
 57. The nano-liquid chromatography method ofclaim 53 further comprising placing the outlet port into fluidcommunication with a different chip inlet by positioning the substrateon the second surface at a different orientation relative to thepackaging jig.
 58. The nano-liquid chromatography method of claim 53wherein the outlet port is placed into fluid communication with thecolumn inlet through an access hole formed through the deposited layeron a chip front side.
 59. The nano-liquid chromatography method of claim53 wherein the outlet port is placed into fluid communication with thecolumn inlet through an access hole formed through the substrate on achip back side.